DNA amplification methods provide a powerful and widely used tool for genomic analysis. Polymerase chain reaction (PCR) methods, for example, permit quantitative analysis to determine DNA copy number, sample source quantitation, and transcription analysis of gene expression. Melting curve analysis is an important tool used to discriminate real amplification products from artifacts, for genotyping, and for mutation scanning. DNA analysis methods allow the detection of single base changes in specific regions of the genome, such as single nucleotide polymorphisms (SNPs). SNP analysis and other techniques facilitate the identification of mutations associated with specific diseases and conditions, such as various cancers, thalassemia, or others.
Statistical assay variations in melt curve data result from system noise in an analysis system, such as the thermal non-uniformity of a thermocycler block in a thermal cycler apparatus. For certain genotyping applications, the melting point shift between samples may be only fractions of a degree. In the case of SNP analysis, the SNP mutations may shift the melting point temperature by no more than 0.2° C.
Accordingly, there is a need in the art for methods of analyzing small differences in melting curves in the presence of the inherent noise of the analysis.